bafilomycin-a1 and Body-Weight

To examine the functional significance and morphological characteristics of starvation-induced autophagy in the adult heart, we made green fluorescent protein-microtubule-associated protein 1-light chain 3 (LC3) transgenic mice starve for up to 3 days. Electron microscopy revealed round, homogenous, electron-dense lipid droplet-like vacuoles that initially appeared in cardiomyocytes as early as 12 hours after starvation; these vacuoles were identified as lysosomes based on cathepsin D-immunopositive reactivity and acid phosphatase activity. The increase in the number of lysosomes depended on the starvation interval; typical autophagolysosomes with intracellular organelles also appeared, and their numbers increased at the later phases of starvation. Myocardial expression of autophagy-related proteins, LC3-II, cathepsin D, and ubiquitin, increased, whereas both myocardial ATP content and starvation integral decreased. Treatment with bafilomycin A1, an autophagy inhibitor, did not affect cardiac function in normally fed mice but significantly depressed cardiac function and caused significant left ventricular dilatation in mice starved for 3 days. The cardiomyocytes were occupied with markedly accumulated lysosomes in starved mice treated with bafilomycin A1, and both the myocardial amino acid content, which was increased during starvation, and the myocardial ATP content were severely decreased, potentially contributing to cardiac dysfunction. The present findings suggest a critical role of autophagy in the maintenance of cardiac function during starvation in the adult.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Autophagy; Body Weight; Cathepsin D; Enzyme Inhibitors; Fluorescent Antibody Technique; Green Fluorescent Proteins; Heart Function Tests; Immunoenzyme Techniques; Lysosomes; Macrolides; Mice; Mice, Transgenic; Microtubule-Associated Proteins; Myocytes, Cardiac; Proton-Translocating ATPases; Rats; Ubiquitin; Vacuoles; Ventricular Dysfunction, Left

The plecomacrolide vacuolar ATPase inhibitors bafilomycin and concanamycin contaminate tuberous vegetables and damage pancreatic islets in mice. The consequences of repeated exposure of adult mice to sub-toxic doses of bafilomycin A1 or concanamycin A was examined by injection of the plecomacrolides on each of five consecutive days. There was a significant reduction in islet size in female C57BL/6j mice (p<0.004 and p<0.0001 respectively). There were no significant differences in fasted insulin levels and beta cell mass between treated and control groups but oral glucose tolerance worsened with increasing age in BALB/c female mice injected with concanamycin A. Streptozotocin reduced glucose tolerance and islet number but not islet size in all strains and sexes. Chronic exposure of C57BL/6j mice to concanamycin A for 16 weeks caused a significant reduction in islet size in both sexes and a significant increase in the spleen weight of female mice (p<0.001). We conclude that repeated exposure to small quantities of vacuolar proton-translocating ATPase inhibitory plecomacrolides reduces islet size and can lead to glucose intolerance, possibly due to impaired maintenance of pancreatic islets. This may lead to earlier progression to beta cell failure and insulin deficiency in those at risk of diabetes.

Topics: Animals; Anti-Bacterial Agents; Blood Glucose; Body Weight; Enzyme Inhibitors; Female; Glucose Tolerance Test; Insulin; Islets of Langerhans; Macrolides; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Organ Size; Proton-Translocating ATPases; Spleen; Streptozocin

We previously isolated berberine from aqueous extracts of tsu-kan-gan, a Kampo formula used for the treatment of osteoporosis. Berberine caused an inhibitory effect on parathyroid hormone (PTH)-stimulated bone resorption in neonatal mouse bone. In this report we describe the inhibitory effect of berberine on the formation of osteoclast-like multinucleated cells (OCLs) in the co-culture of mouse osteoblastic cells and bone marrow cells in the presence of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], PTH and interleukin-1alpha (IL-1alpha). Berberine dose-dependently inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive OCLs induced by 1alpha25(OH)2D3, PTH and IL-1alpha. We prepared OCLs in the co-culture of osteoblastic cells and bone marrow cells. The effect of berberine on pit formation by OCLs was examined using dentin slices. As OCLs are terminally differentiated multinucleated cells, the survival of OCLs affects the bone-resorbing activity of OCLs. This prompted us to count the number of TRAP-positive OCLs on the slices. Berberine dose-dependently inhibited pit formation and caused a decrease in the number of TRAP-positive OCLs. Calcitonin (CT) inhibited pit formation without affecting the number of OCLs. Berberine accelerated the cell death in OCLs cultivated on a culture plate, but CT did not affect the cell death of OCLs. This suggests that the decrease in the number of OCLs on dentin slices may be due to apoptotic cell death in OCLs. In fact, Hoechst 33258 staining revealed that the treatment of OCLs with berberine resulted in condensed nuclei and a decrease in cell size. Oral administration of the berberine (30 and 50 mg/kg/d) to ovariectomized rats prevented a decrease in bone mineral density (BMD) of the lumbar vertebra without affecting the weight of the uterus and plasma concentration of estradiol. These results suggested that berberine prevented a decrease in BMD in vivo by inhibiting osteoclastic bone resorption.

Topics: Animals; Anti-Bacterial Agents; Apoptosis; Berberine; Body Weight; Bone Density; Bone Resorption; Calcitonin; Calcitriol; Drugs, Chinese Herbal; Female; Interleukin-1; Macrolides; Male; Mice; Organ Size; Osteoclasts; Ovariectomy; Parathyroid Hormone; Rats; Rats, Wistar; Uterus

Adaptive increases in renal bicarbonate reabsorption occur in response to acute increases in filtered bicarbonate (FLHCO3). In a previous study, we showed that an increase in FLHCO3 induced by plasma volume expansion increased the Vmax for Na+/H+ exchange activity in renal cortical brush border membrane vesicles (BBMV), providing a potential mechanism for the adaptive increase in HCO3- reabsorption. The present studies were undertaken to determine whether the increase in FLHCO3 induced by plasma expansion also stimulates the other major H+ transporter in cortical BBMV, the H(+)-ATPase. H(+)-ATPase activity was assessed in BBMV obtained from hydropenic and plasma expanded Munich-Wistar rats, using a NADH-linked ATPase assay. H(+)-ATPase activity was measured as the ouabain and oligomycin-insensitive, bafilomycin A1-sensitive component of total ATPase activity. Acute plasma expansion doubled single nephron FLHCO3, and this change was associated with a 64% increase in the Vmax for H(+)-ATPase activity, with no change in apparent Km. The Vmax for H(+)-ATPase activity correlated directly with whole kidney GFR and FLHCO3 (r = 0.68 and 0.72, respectively), and with single nephron GFR and FLHCO3 (r = 0.76 and 0.80, respectively). Thus, the mechanism for the adaptive increase in proximal tubular HCO3- reabsorption that occurs in response to acute increases in FLHCO3 appears to be related to increased activity of both H(+)-ATPase and Na+/H+ exchange in the apical membrane of the proximal tubule epithelium.

Topics: Absorption; Adaptation, Physiological; Adenosine Triphosphate; Animals; Anti-Bacterial Agents; Bicarbonates; Body Weight; Enzyme Inhibitors; Isotonic Solutions; Kidney Tubules, Proximal; Kinetics; Macrolides; Male; Microvilli; Oligomycins; Ouabain; Plasma Substitutes; Proton-Translocating ATPases; Rats; Rats, Wistar; Ringer's Lactate; Sodium-Hydrogen Exchangers; Ultrafiltration